5-Fluorouracil derivatives

ABSTRACT

Novel compounds comprising 5-fluorouracil or 5-fluorouridine covalently linked to 5-ethynyluracil, 5-ethynyluridine or 5-propynyluracil and pharmaceutical compositions comprising such compounds are disclosed.

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application No.PCT/GB94/02428 filed Nov. 4, 1994 which claims priority from GB9322795.7filed Nov. 5, 1993.

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application No.PCT/GB94/02428 filed Nov. 4, 1994 which claims priority from GB9322795.7filed Nov. 5, 1993.

The present invention relates to compounds which comprise certain enzymeinactivators which are useful in medicine, particularly cancerchemotherapy, covalently linked to antineoplastic agents, particulary5-fluorouracil (5-FU).

5-Fluorouracil has been used in cancer chemotherapy since 1957.Sensitive tumours include breast cancer, gastrointestinal malignancies,and cancers of the head and neck; 5-fluorouracil is also used as aradiation sensitiser. 5-Fluorouracil is metabolised rapidly in the liver(half-life between 8 and 20 minutes) by the enzyme dihydrothymidinedehydrogenase (uracil reductase). It has been reported (Cancer Research46, 1094, 1986) that 5-(2-bromovinyl)-uracil (BVU) is an inhibitor ofdihydrothymidine dehydrogenase which both retards the metabolism of5-fluorouracil and enhances its antitumour activity. It has beenreported that 5-(2-bromovinyl)-2'-deoxyuridine (which is metabolised invivo to BVU) enhances the antitumour activity of 5-fluorouracil and5-deoxy-5-fluorouridine, a prodrug of 5-fluorouracil (BiochemicalPharmacology 38; 2885, 1989).

W092/04901 discloses 5-substituted uracil derivatives which are usefulas inactivators of uracil reductase; they increase the level andhalf-life of 5-fluorouracil in plasma and enhance the activity of5-fluorouracil. These derivatives also reduce the normally encounteredvariations of 5-fluorouracil plasma levels between subjects.5-ethynyluracil is 100-fold more potent than BVU as an inactivator ofuracil reductase.

It has now been found that useful compounds can be produced bycovalently linking a uracil reductase inactivator moiety with a5-fluorouracil moiety or a prodrug thereof.

Therefore a first aspect the present invention provides a compound ofthe formula:

    X-L-Y                                                      (I)

wherein X represents a uracil reductase inactivator or prodrug thereof,Y represents 5-fluorouracil or a prodrug thereof, and L is a linkinggroup covalently linked to both X and Y. Preferably X is a 5-substitutedor 5,6-dihydro-5-substituted uracil derivative, the 5-substituent beingbromo, iodo, cyano, halo-substituted C₁₋₄ alkyl, C₂₋₆ alkenyl, halosubstituted C₂₋₆ alkenyl group, a C₂₋₆ alkynyl group, halo substitutedC₂₋₆ alkynyl group.

These compounds when administered to a subject, for example a mammalsuch as a human, provide both a uracil reductase inactivator as well asthe 5-fluorouracil antineoplastic agent itself. Thus, only a singleactive agent need be administered to a subject.

In addition, these compounds have the advantage that they can beadministered orally. 5-Fluorouracil cannot normally be administeredorally, as it is destroyed by uracil reductase in the gastrointestinaltract. A single agent, which provides 5-FU and which can be administeredorally, is now provided.

By a C₂₋₆ alkenyl or C₂₋₆ alkynyl group is meant a straight or branchedchain alkenyl or alkynyl group, the latter including an alkenyl oralkynyl group substituted by a C₂₋₆ cycloalkyl group.

The halogen substituent on the alkenyl or alkynyl group is preferablybromo, chloro or iodo. Halo-substituted ethenyl and ethynyl groups areparticularly preferred. Usually only one halo substituent will bepresent. Preferred halo-substituted alkenyl groups are substituted inthe 1-position.

The uracil derivative moiety is preferably one wherein the 5-substituentis a C₂₋₆ alkynyl group (optionally halo-substituted), conveniently aC₂₋₄ alkynyl group and preferably an ethynyl or propynyl group. Inpreferred 1-halo-alkenyl and alkynyl derivatives the multiple bond is inthe 1-position. Particularly preferred uracil derivatives which formpart of the compounds as hereinbefore defined are 5-ethynyluracil and5-propynyluracil. Other such inactivators include:

5-cyanouracil

5-bromoethynyluracil

5-(1-chlorovinyl) uracil

5-iodouracil

5-hex-1-ynyluracil

5-vinyluracil

5-trifluoromethyluracil

5-bromouracil

Prodrugs of 5-fluorouracil are compounds which are metabolised in vivoto 5-fluorouracil and include 5-fluorouridine, 5-fluoro-2-deoxyuridine,5-fluoro-2-deoxycytidine, 5'-deoxy-5-fluorouridine,1-(2-tetrahydrofuranyl)-5-fluorouracil and 1-C₁₋₈alkylcarbamoyl-5-fluorouracil derivatives.

Prodrugs of the uracil reductase inactivators are compounds which aremetabolised in vivo to the corresponding uracil reductase inactivator.

Such prodrugs include nucleoside analogues which contain a nucleobasecorresponding to the above 5-substituted uracil compounds. For examplenucleoside derivatives containing a ribose, 2'-deoxy-ribose, arabinoseor other cleavable sugar portion, which may contain a 2' or3'-substituent such as halo, e.g. chloro or fluoro; alkoxy; amino orthio. Specific example of such nucleoside derivatives are1-(6-O-arabinofuranosyl)-5-ethynyluracil and1-(6-O-arabinofuranosyl)-5-prop-1-ynyluracil. Other uracil derivativeprodrugs include 5-ethynyl-2-pyrimnidinone.

The nature of the linking group, L, should be such that the activecomponents are released once the compound has been administered to thesubject.

Thus, breakdown of a compound of the invention will result in release ofboth 5-fluorouracil and the uracil reductase inactivator, or prodrugsthereof.

In a preferred embodiment both the 5-fluorouracil moiety and the uracilreductase inactivator moiety are presented in the form of nucleosides,these then being linked such that upon administration to the subject thelinkage breaks down releasing the active components.

Suitable sugar moieties which together with 5-fluorouracil and theuracil reductase inactivator can form such nucleosides include ribose,2'-deoxyribose, arabinose and 5'-Cl-2'-deoxyribose.

Examples of suitable linking groups, L, include succinate groups,pyrophosphate groups, tartrate groups, mono-, di- or tri- phosphategroups and amide groups, with succinate and phosphate groups in generalbeing particularly suitable.

Examples of preferred compounds of the invention include:

5-ethynyl-5"-fluoro-3',3'"-O-succinylbis-(1-(2-deoxy-β-D-erythropentofuranosyl)uracil);

5-ethynyluridine-5-fluoro-1-(β-D-arabinofuranosyl)uracil succinic acid5', 2'-diester;

5-fluoro-5"-(1-propynyl)-2', 2'"-O-succinylbis(1-β-D-arabinofuranosyluracil); and

5'-chloro-2'-5'-dideoxy-5-fluorouridine 2'"-deoxy-5"-ethynyluridine(3'-5'")diphosphate.

In general a suitable dose of a compound as hereinbefore described willbe such as to provide a dose of 5-fluorouracil or a prodrug thereof inthe range of 0.1 to 1000 mg per kilogram body weight of the recipientper day, preferably in the range of 0.1 to 200 mg per kilogram bodyweight per day. Most preferable is a dose in the range of 0.1 to 50 mgper kilogram body weight per day. For the uracil derivative component asuitable dose is in the range of 0.01 to 50 mg per kilogram body weightof the recipient per day, particularly 0.01 to 10 mg/kg. More preferablythe dose should be such as to provide 0.01 to 0.4 mg per kilogram bodyweight per day as uracil derivative. Accordingly, a suitable dose of acompound as hereinbefore described will be in the range of 0.04 to 4000mg per kilogram body weight per day, preferably in the range of 0.04 to800 mg per kilogram body weight per day and most preferably in the rangeof 0.4 to 200 mg per kilogram body weight per day.

Given the nature of the compound it will be seen that it is possible, byincluding further linking groups, to increase the number of5-fluorouracil moieties, or prodrugs thereof such that upon breakdown ofthe compound the dosages of 5-fluorouracil, or prodrug thereof, anduracil derivative fall within the abovementioned ranges.

Therefore the invention also provides compounds of the formulae (II) and(III):

X-(L-Y)n (II) and X-L-(Y)n (III)

Wherein X, L, and Y are as hereinbefore defined and n is at least 2.Preferably n is in the range 2-10.

For brevity, the term "a compound as hereinbefore defined" is usedhereinafter to describe compounds of formula (I), (II) and (III).

In a further aspect the present invention provides a compound ashereinbefore defined for use in medicine, in particular for use incancer chemotherapy.

In a still further aspect the present invention provides the use of acompound as hereinbefore defined in the manufacture of a medicament foruse in cancer chemotherapy. The medicament may also be useful for thetreatment of psoriasis, rheumatoid arthritis, or human papilloma virusinfections.

In a further aspect the present invention provides a method for thetreatment of cancer in a mammal comprising administering an effectiveamount of a compound as hereinbefore defined to said mammal.Alternatively, there is provided a method of reducing or inhibiting thetumour burden comprising administering to a mammal in need thereof aneffective amount of a compound as hereinbefore defined.

In other aspects the invention further provides:

a) A method for the treatment or prophylaxis of psoriasis, rheumatoidarthritis or human papilloma virus infection which comprisesadministering an effective amount of a compound as hereinbefore definedto a mammal;

b) A method of reducing the toxicity and/or potentiating the efficacyand/or increasing the therapeutic index of 5-FU which comprisesadministering an effective amount of a compound as hereinbefore definedto a mammal;

c) A method of generating 5-ethynyluracil in a mammal which comprisesadministering a compound as hereinbefore defined wherein the uracilreductase inactivator is 5-ethynyluracil or a prodrug thereof, to amammal; and

d) A method of generating 5-fluorouracil in a mammal which comprisesadministering a compound as hereinbefore defined to a mammal.

Preferably the mammal is a human.

The required dose of the compound may be administered in unit dosageforms. The desired dose is preferably presented as one, two or moresub-doses administered at appropriate intervals. These sub-doses may beadministered in unit dosage forms containing for example 1 to 200 mg ofthe compound.

The compound is preferably administered in the form of a pharmaceuticalcomposition. Thus, in a further aspect the present invention provides apharmaceutical composition comprising a compound as hereinbefore definedtogether with at least one pharmaceutically acceptable carrier orexcipient, and optionally one or more other therapeutic ingredients.

Each carrier or excipient must be "pharmaceutically acceptable" in thesense of being compatible with the other ingredients of the compositionand riot injurious to the patient. Compositions include those adaptedfor oral, rectal, nasal, topical (including buccal, transdermal andsublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Compositions of the present invention adapted for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granule; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Oral administration is thepreferred route.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to providecontrolled release of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile.

A capsule may be made by filling a loose or compressed powder on anappropriate filling machine, optionally with one or more additives.Examples of suitable additives include binders such as povidone;gelatin, lubricants, inert diluents and disintergrants as for tablets.Capsules may also be formulated to contain pellets or discrete sub-unitsto provide slow or controlled release of the active ingredient. This canbe achieved by extruding and spheronising a wet mixture of the drug plusan extrusion aid (for example microcrystalline cellulose) plus a diluentsuch as lactose. The spheroids thus produced can be coated with asimi-permeable membrane (for example ethyl cellulose, Eudragit WE30D) toproduce sustained release properties.

Compositions for topical administration in the mouth include lozengescomprising the active ingredient in a flavoured base, usually sucroseand acacia or tragacanth; pastilies comprising the active ingredient inan inert base such as gellatin or glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Compositions for vaginal administration may be presented as pessaries,tampons creams, gels, pastes, foams or spray formulations containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Compositions for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render thecomposition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multidose sealed containers, for example, ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemproaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as hereinabove recited, or an appropriate fractionthereof, of an active ingredient.

Compounds of formula (I) are novel and accordingly a process for thepreparation of these compounds provides a further aspect of the presentinvention.

Compounds of formula (1) may be prepared by reacting a uracil reductaseinactivator or prodrug thereof, or a monoprotected derivative of auracil reductase inactivator or prodrug thereof, with a compound L'-Ywherein Y is as hereinbefore defined or a protected derivative thereofand L' is a group capable of reacting with the uracil reductaseinhibitor to form the linking group L, followed by deprotection wherenecessary. Suitable protecting groups will be known by one skilled inthe art, and include tert-butyldimethylsilyl chloride,trimethylsilylacetylene or p-anisylchlorodiphenylmethane(methoxytrityl).

The above coupling reaction may be carried out in a polar aproticsolvent, such as pyridine, in the presence of a coupling reagent, forexample dicyclohexylcarbodiimide (DCC) or dimethylaminopyridine (DMAP).The reaction is carried out at a non-extreme temperature of -5° to 100°C., most suitably room temperature. Methods for the removal of theprotecting groups may be carried out by methods known in the art, forexample ion exchange using Dowex 50W-X8 H⁺ ! in methanol can be used toremove the trityl groups (C. Malange, Chem. Ind. (1987) 856) and silylprotecting groups may be removed using tetraethyl ammonium fluoride inacetonitrile (S. Hanessian et al. Can. J. Chem. (1975) 53, 2975).

The group L' may be attached to the group Y by the reaction of acompound capable of creating a linking group with the compound Y in thepresence of a suitable polar aprotic solvent, for example1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMTHP). The reactionis carried out at a non-extreme temperature of -25° to 100° C., mostsuitably -5° C. to room temperature. Compounds capable of forminglinking groups include succinic anhydride, phosphorous oxychloride. Sucha process for the preparation of a compound of formula (I) wherein X andY are both nucleoside prodrugs, may more clearly be understood byreference to Scheme 1.

To prepare compounds wherein X and Y are joined via a symmetricallinker, both X and Y may be reacted with a linking group, and the tworesulting compounds combined to give a compound of formula (I) asdescribed in Scheme 2 for the preparation of a compound of formula (I)wherein X and Y are both nucleoside prodrugs.

Intermediates of formula L'-Y are novel and accordingly provide afurther aspect of the present invention. ##STR1##

The invention will now be described with reference to, but not limitedby, the following examples:

EXAMPLE 1 Preparation of 5-ethynyl-5"-fluoro-3,3"-O-succinylbis(1-(2-deoxy-β-D-erythropento furanosyl)uracil)

(a) 5'-O-(tert-Butyldimethylsilyl)-2'-deoxy-5-fluorouridine5-Fluoro-2'-deoxyuridine (United States Biochemical Corporation,Cleveland, Ohio 44120) (6.00 g, 24.4 mmol) and 3.65 g (5.36 mmol)imidazole (Aldrich Chemical Company, Milwaukee, Wis. 53233) weredissolved in 25 ml anhydrous DMF in a flame dried round bottom flask.4.0 g (26.8 mmol) of tert-Butyldimethylsilyl chloride (Aldrich) wereadded all at once, the flask was capped with a serum stopper and thesolution was magnetically stirred over 3 days. After this time, 100 mlof saturated aqueous sodium bicarbonate was poured into the solution andthe mixture was extracted with 2×200 ml ethyl acetate. The ethyl acetatelayer was washed with 200 ml water, dried over magnesium sulphate andthe solvent was removed on the rotary evaporator. The resulting residuewas applied to a column (4.5×30 cm) packed with silica gel (350 g,230-400 mesh) in dichloromethane. Under medium air pressure (9-12 psi)the column was first eluted with IL of 1% methanol in dichloromethane;successively eluted with 1 L of 1.5% methanol in dichloromethane, then 3L of 2% methanol in dichloromethane and finally eluted with 3.5 L of 3%methanol in dichloromethane. Product fractions were pooled and thesolvent evaporated to give 6.9 g (19.1 mmol) of the desired product as awhite powder; Rf 0.34 (silica, 9:1 chloroform: methanol). ¹ H NMR(CDCl₃) δ8.85 (bs,1H, NH), 8.04 (d,1H, H6, J_(F),H =6.4 Hz), 6.38-6.35(dd overlapping, 1H, H1'), 4.50-4.46 (m, 1H, H3',) 4.08-4.06 (m, 1H,H4'), 3.97-3.92 (dd, 1H, H5, J=2.4 Hz, J=11.5 Hz), 3.86-3.81(dd, 1H,H5", J=2.0 Hz, J=11.4 Hz) 2.54-2.38 (m, 1H, H2"), 2.18-2.11(m,1H,H2"),0.93 (s, 9H, tert-butyl), 0.15 (s, 6H, silyl methyls).

(b)5'-O-(tert-Butyldimethylsilyl)-3'-O-(3-Carboxypropionyl)-2'-deoxy-5-fluorouridine

4.0 g (11.0 mmol) of5'-O-(tert-Butyldimethylsilyl)-2'-deoxy-5-fluorouridine was dried bytwice dissolving the nucleoside in 25 ml of anhydrous pyridine in around bottom flask and evaporating the pyridine on a rotary evaporator.To the thus dried protected nucleoside re-dissolved in 20 ml pyridinewas added 0.54 g (4.4 mmol) of 4-dimethylaminopyridine all at once and0.84 g (8.4 mmol) succinic anhydride (Aldrich Chemical Company,Milwaukee, Wis. 53233) in three portions over 90 min. After stirring for2.5 hrs, another equal portion of 4-dimethylaminopyridine was added. Theflask was sealed with a serum stopper and the mixture was allowed tostir overnight. After this time, pyridine was removed on the rotaryevaporator by co-evaporation with 4×50 ml toluene to give a tan foam.The foam was dissolved in 100 ml dichloromethane and extracted with3×100 ml cold aqueous 10% citric acid, followed by 100 ml water. Thedichloromethane layer was dried over magnesium sulphate, filtered, andthe solvent was removed on a rotary evaporator to give 3.9 g of a tanfoam. This foam was dissolved in a minimal amount of dichloromethane andapplied to a column (4.5×30 cm) packed with silica gel (350 g, 230-400mesh) in dichloromethane. Under medium air pressure (9-12 psi) thecolumn was first eluted with 1 L of chloroform followed by elution with1 L of 1% methanol in chloroform. The percentage of methanol wasincreased 1% for each successive litre. The product was eluted intofractions between 2% and 10% methanol in chloroform. Product fractionswere pooled and the solvent was removed on the rotary evaporator to give2.0 g (4.3 mmol) of a white solid: m.p. 133.4°-135.3° C.

(c) 5'-O-(tert-Butyldimethylsilyl)-2'-deoxy-5-iodouridine

5-Iodo-2'-deoxyuridine (United States Biochemical Corporation, ClevelandOhio 44120) (10.0 g, 28.2 mmol) and 4.23 g (62.1 mmol) of imidazole(Aldrich Chemical Company, Milwaukee, Wis. 53233) were dissolved in 50ml anhydrous dimethylformamide (DMF) in a flame-dried round bottomflask. 4.7 g (31.0 mmol) of tert-Butyldimethylsilylchloride (Aldrich)were added all at once; the flask was capped with a serum stopper andthe solution was magnetically stirred for 11 days. After this period oftime, the reaction mixture was poured into 300 ml aqueous saturatedsodium bicarbonate and extracted with 4×150 ml ethyl acetate. The ethylacetate extraction's were pooled, dried over magnesium sulphate,filtered and the solvent was removed on a rotary evaporator. The residuewas applied to a column (4.5×30 cm) packed with silica gel (350 g,230-400 mesh) in dichloromethane. Under medium air pressure (9-12 psi)the column was first eluted with 2 L of dichloromethane, followed bystep-elution with 2 L of 1% methanol in dichloromethane, 2 L of 2%methanol in dichloromethane and finally 1 L of 3% methanol indichloromethane. Pure product fraction were pooled and solvent removedon a rotary evaporator to give 8.8 g (18.8 mmol) of a white powder: Rf0.30 (9:1 chloroform: methanol). ¹ H NMR (300 MHz, CDCl₃) δ8.30-8.20(bs,1H, NH), 8.10 (s,1H, H6), 6.32-6.27 (dd overlapping, 1H, H1')4.49-4.46 (m, 1H, H3'), 4.10-4.07 (m,1H, H4'), 3.94-3.89 (dd, 1H, H5",J_(5"), 5' =11.5 Hz, J_(5"), 4' =2.6 Hz), 3.85-3.81 (dd, 1H, H5', J₅',5"=11.5 Hz, J_(5'),4' =2.3 Hz), 2.45-2.42 (m, 1H, H2"), 2.41-2.38 (m,1H,H2'), 0.96 (S,9H, tert-butyl), 0.93 (s, 3H, silyl methyl), 0.92 (s,3H, silyl methyl).

(d)1-(5-O-(tert-Butyldimethylsilyl)-2-deoxy-β-D-erythro-pentofuranosyl-5-(2-(trimethylsilyl)ethynyl)uracil)

To a solution of 5.0 g (10.7 mmol) of5'-O-(tert-butyldimethylsilyl)-2'-deoxy-5-iodouridine, 0.62 g (3.3 mmol)copper (I) iodide, and 3.3 g (4.6 ml, 32.8 mmol) triethylamine in 50 mldimethylformamide (DMF) under a nitrogen atmosphere was added 4.8 g (7.0ml, 49.3 mmol) of trimethylsilylacetylene and 1.9 g (1.6 mmol) oftetrakis(triphenylphosphine)palladium(0) (Aldrich Chemical Company,Milwaukee, Wis. 53233). The dark mixture was magnetically stirred atroom temperature overnight. DMF was removed in vacuo on a rotaryevaporator and the dark oily residue was dissolved in 100 ml chloroformand extracted with 2×150 ml of aqueous 5% ethylenediamine tetra aceticacid disodium salt, followed by 1×150 ml water. The chloroform layer wasdried over magnesium sulphate, filtered and the solvent removed on arotary evaporator. The residue was dissolved in a minimal amount ofchloroform and applied to a column (4.5×30 cm) packed with silica gel(350 g, 230-400 mesh) in chloroform. Under medium air pressure (9-12psi) the column was eluted with 2 L of chloroform followed by 3.5 L of1% methanol in chloroform to elute the desired product. After removal ofthe solvent on a rotary evaporator, 2.7 g (6.2 mmol) of product wasobtained as a tan solid: Rf 0.51 (silica, 9:1 chloroform: methanol). ¹ HNMR (300 MHz, CDCl₃) δ8.15-8.10(bs,1H,NH),7.97 (s,1H, H6), 6.34-6.29 (ddoverlapping, 1H, H1'), 4.51-4.48 (m, 1H, H3') 4.10-4.08 (m, 1H, H4'),3.95-3.90 (dd,1H, H5", J_(5"),5' =11.4 Hz, J_(5"),4' =2.6 Hz), 3.85-3.80(dd 1H, H5', J_(5'),5" =11.4 Hz, J_(5'),4' =2.1 Hz), 2.41-2.37 (m, 1HH2"), 2.16-2.11 (m, 1H H2"), 1.86 (s, 9H, C(CH₃)₃), 0.20 (s, 9H,acetylenic Si(CH₃)₃), 0.15 (s, 3, Silyl methyl), 0.13 (s, 3H,silylmethyl).

(e) 5-Fluoro-5"-(2-(trimethylsilyl)ethynyl-3',3"'-O-succinylbis-(1-(2-deoxy-5-O-(tert-butyldimethylsilyl)-β-D-erythro-pentofuranosyl)uracil)

In a previously flame dried round bottom flask was dissolved 0.51 g (1.1mmol) of1-(5-O-(tert-butyldimethylsilyl)-2-deoxy-β-D-erythro-pentofuranosyl-5-(2-(trimethylsilyl)ethynyl)uracil and 0.49 g (1.1 mmol) of5'-O-(tert-butyldimethylsilyl)-3'-O-(3'-carboxypropionyl)-2'-deoxy-5-fluorouridinein 5 ml anhydrous pyridine. To this solution was added 0.46 g (2.2 mmolof dicyclohexylcarbodiimide (Aldrich Chemical Company, Milwaukee, Wis.53233) and the solution was allowed to magnetically stir overnight. Thefollowing day, pyridine was removed on the rotary evaporator and theorange-brown syrup was resuspended in ice-cold ethyl acetate.Dicyclohexylurea was removed by vacuum filtration and the ethyl acetatewas subsequently removed on the rotary evaporator. The residue wasre-dissolved in a minimal amount of dichloromethane and applied to acolumn (4.5×30 cm) packed with silica gel (350 g, 230-400 mesh) indichloromethane. Under medium air pressure (9-12 psi) the column waseluted with 2 L of dichloromethane followed by 4 L of 1.5% methanol indichloromethane for elution of an impure product. The product wasre-chromatographed across 100 g silica packed in hexanes under mediumair pressure and eluted by an increasing step-gradient from 5 to 50%ethyl acetate in hexanes. After removal of solvent on a rotaryevaporator, 0.60 g (0.68 mmol) of the product was obtained as a whitepowder: R_(f) 0.54 (silica, 9:1 chloroform: methanol); m.p. 139.8° C.,resolidifies to melt at 208°-210° C.

(f)5-Ethynyl-5"-fluoro-3',3"-O-succinylbis-(1-(2-deoxy-β-D-erythro-pentofuranosyl)uracil)

0.90 g (1.0 mmol) of5-Fluoro-5"-(2-trimethylsilyl)ethynyl-3',3'"-O-succinylbis(1-(2-deoxy-5-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranosyl)uracil)was treated with 3.3 equivalents of tetraethyl ammonium fluoride hydrate(Aldrich Chemical Company, Milwaukee, Wis. 53233) in 25 ml acetonitrileand allowed to magnetically stir at ambient temperature overnight.

The following day, an additional equivalent of the fluoride reagent wasadded and the solution was allowed to stir for several more hours. Thesolution was then directly applied to a 100 g silica gel (230-400 mesh)column (2.5×30 cm) packed in dichloromethane. The column was eluted with500 ml of dichloromethane, followed by a step-gradient of 1 to 5%methanol in dichloromethane. Overnight, the fractions precipitated outof the 5% methanol fractions to give 156 mg of analytically pure productafter filtration and drying. The combined mother liquors were combinedand evaporated to give an additional 270 mg of product after drying fora total of 0.426 g (0.73 mmol): m.p. 229.5°-230.6° C. ¹ H NMR (DMSO-d₆)δ11.9-11.6 (bs, 2H, N3H & N3"H), 8.29 (s, 1H, H6), 8.21 (d, 1H, H6",J_(F), H =7.1 Hz), 6.16-6.12 (m, 2H, H1' & H1'"), 5.33-5.30 (m, 2H, H3'& H3'"), 5.29-5.24 (m, 2H, 5'OH & 5'"OH), 4.14 (s, 1H, acetylenic H),4.04-4.01 (m, 2H, H4' & H4'"), 3.65-3.63 (m, 4H<H5' & H5'"), 2.65 (s,4H, succinate), 2.33-2.26(m, 4H, H2' & H2'").

EXAMPLE 2 Preparation of5-ethynyl-5"-fluoro-3',3"-O-succinylbis(1-(2-deoxy-β-D-erythropentofuranosyl)uracil)

(a) 2'-Deoxy-5-fluoro-5'-O-(4-methoxytrityl)uridine

5-Fluoro-2'-deoxyuridine (United States Biochemical Corporation,Cleveland, Ohio 44120) (1.0 g, 4.1 mmol) was twice suspended in 30 mlanhydrous pyridine in a round bottom flask and the pyridine removed on arotary evaporator at 50° C. and oil pump vacuum. The deoxyribonucleosidethus dried was re-dissolved in 41 ml pyridine and cooled to ca. 5° C. inan ice-water bath. The solution was magnetically stirred. To thesolution was added 0.06 equivalents (30 mg, 0.24 mmol)4-dimethylaminopyridine and 1.4 equivalents triethylamine (0.8 ml, 5.7mmol) all at once. This was followed by the addition of 1.2 equivalents(1.5 g, 4.9 mmol) p-anisylchlorodiphenylmethane (Aldrich ChemicalCompany, Milwaukee, Wis. 53233) in one-quarter portions every 45 min.After the final addition of p-anisylchlorodiphenylmethane, the ice-bathwas allowed to gradually warm to ambient temperature. The reaction wassealed with a serum stopper and magnetically stirred overnight. Afterthis period an additional 350 mg of p-anisylchlorodiphenylmethane wasadded and the reaction was allowed to stir for another 8 hrs. Thereaction was then poured into 100 ml water in a separating funnel andextracted with 4×100 ml diethyl ether. Diethyl ether was removed on arotary evaporator. 2.4 g of crude material was dissolved in a minimalamount of dichloromethane and applied to a column (2.5×30 cm) packedwith silica gel (100 g, 230-400 mesh) in dichloromethane. Under mediumair pressure (9-12 psi) the column was first eluted with 2 L ofdichloromethane followed by 1 L of 3% methanol in dichloromethane toelute the product. After evaporation of solvent, the product wasobtained as a white foam (1.8 g, 3.2 mmol): Rf 0.42 (silica, 9:1chloroform: methanol). ¹ H NMR (300 MHz, DMSO-d₆) δ 11.87 (s, 1H, NH)7.88 (d, 1H, H6, J_(F), H=6.85 Hz), 7.42-7.23 (m, 10H, Ar H), 6.90 (d,2H, Ar H, J=8.89 Hz), 6.14-6.12 (overlapping dd, 1H, H2'), 5.34 (d, 1H,3'OH, J=4.57 Hz), 4.27 (m, 1H, H3'), 3.88 (m,1H,H4') 3.75 (s, 3H, OCH₃)3.34-3.25 (m, 1H, H5'), 3.16-3.12 (m, 1H, H5"), 2.26-2.16 (m, 2H, H2" &H2').

(b) 2'-Deoxy-5-iodo-5'-O-(4-Methoxytrityl)uridine 5-Iodo-2'-deoxyuridine(United States Biochemical Corporation, Cleveland, Ohio 44120) (1.0 g,2.8 mmol) was twice suspended in 30 ml anhydrous pyridine in a roundbottom flask and the pyridine removed on a rotary evaporator at 50° C.and oil pump vacuum. The deoxynucleoside thus dried was re-dissolved in28 ml pyridine and cooled to ca 5° C. in an ice-water bath. The solutionwas magnetically stirred. To the solution was added 0.06 equivalents (20mg, 0.16 mmol) 4 -dimethylaminopyridine and 1.4 equivalents (0.55 ml,4.0 mmol) triethylamine all at once followed by the addition of 1.2equivalents (1.0 g, 3.4 mmol) of p-anisylchlorodiphenylmethane (AldrichChemical Company, Milwaukee, Wis. 53233) in one-quarter portions every45 min. After the addition of the final portion ofp-anisylchlorodiphenylmethane, the ice bath was allowed to graduallywarm to ambient temperature. The reaction was sealed with a serumstopper and magnetically stirred overnight. After this period, anadditional 250 mg of p-anisylchlorodiphenyl methane was added and thereaction was allowed to stir for an additional 8 hrs. The reaction wasthen poured into 100 ml water in a separating funnel and extracted with4×100 ml diethyl ether. The diethyl ether extracts were cooled and thesolvent was removed on a rotary evaporator. 2.4 g of crude material wasdissolved in a minimal amount of dichloromethane and applied to a column(2.5×30 cm) packed with silica gel (100 g, 230-400 mesh indichloromethane). Under medium air pressure (9-12 psi), the column wasfirst eluted with 1 L of dichloromethane, then with 1 L of 1% methanolin dichloromethane and finally with 1 L of 5% methanol indichloromethane to elute the product. After evaporation of solvent, theproduct was obtained as an off-white foam (1.3 g, 1.9 mmol): R_(f) 0.48(silica, 9:1 chloroform:methanol). ¹ H NMR (300 MHz, DMSO-d₆) δ 11.75(s, 1H, NH), 8.02 (s, 1H, H6), 7.43-7.23 (m, 10H, Ar H), 6.91 (d, 2H, ArH, J=8.89 Hz), 6.13-6.08 (overlapping dd, 1H, H1'), 5.32 (d,1H, 3'OH,J=4.57 Hz) 4.24 (m, 1H, H3'), 3.91 (m, 1H, H4'), 3.75 (s,3H,OCH₃),3.34-3.17 (m, 2H, H5' & H5"), 2.26-2.17 (m, 2H, H2" & H2).

(c) 2'-Deoxy-5-ethynyl-5'-O-(4-methoxytrityl)uridine

2'-Deoxy-5-iodo-5'-O-(4-methoxytrityl)uridine (1.2 g, 1.8 mmol) wasdissolved in 6 ml of anhydrous N,N-dimethylformamide (DMF) and thesolution was vigorously deoxygenated with nitrogen for 30 min. To themagnetically stirred solution was added 142 mg (0.20 mmol)bis(triphenylphosphine)palladium(II) chloride, 76 mg (0.40 mmol) copper(I) iodide, 0.53 ml (5.40 mmol) of trimethylsilylacetylene and 0.56 ml(4.0 mmol) of triethylamine (Aldrich Chemical Company, Milwaukee, Wis.53233). The following day identical portions ofbis(triphenylphosphine)palladium(II)chloride, copper (I) iodide,trimethylsilylacetylene and triethylamine were added. The reaction wasallowed to stir for an additional 5 hrs before being poured into 100 mlethyl acetate in a separating funnel. The ethyl acetate solution wasextracted with 3×30 ml aqueous 5% ethylenediamine tetra acetic aciddisodium salt and washed with 1×30 ml water. Ethyl acetate was removedon the rotary evaporator to give a black foam. This foam was dissolvedin 5 ml of acetonitrile and 222 mg (1.5 mmol) of tetraethyl ammoniumfluoride hydrate (Aldrich Chemical Company, Milwaukee, Wis. 53233) wasadded and the solution was magnetically stirred for 1 hr. After thisperiod the reaction mixture was loaded directly onto a column (2.5 C 30cm) packed with silica gel (100 g, 230-400 mesh) in chloroform. Undermedium air pressure (9-12 psi) the column was first eluted with 1 L ofchloroform, then 1 L of 1% methanol in chloroform and finally with 1 Lof 2.5% methanol in chloroform to elute the product. The appropriateproduct fractions were combined and solvent was removed on a rotaryevaporator to 769 mg (1.5 mmol) of a light brown foam: Rf 0.23 (silica,95:5 chloroform: methanol) ; IR (1% KBr) acetylenic carbon-carbonstretch 2110 cm⁻¹ ; MS (EI, Cl,) 524 (M). ¹ H NMR (300 MHz, DMSO - d₆) δ11.69 (s, 1H, NH), 7.94 (s, 1H, H6), 7.41-7.21 (m, 10H, Ar H), 6.90 (d,2H, Ar H, J=8.9 Hz), 6.11-6.07 (overlapping dd, 1H, H1'), 5.30 (d, 1H,3' OH, J=4.6 Hz), 4.24 (m, 1H, H4'), 3.74 (s, 3H OCH₃), 3.41-3.11 (m,overlapping water peak, H5' & H5"), 2.27-2.18 (m, 2H, H2" & H2').

(d)5-Ethynyl-5"-fluoro-3',3'"-O-succinylbis(1-(2-deoxy-5-0-(4-methoxytrityl)-β-D-erythro-pentofuranosyl)uracil)

To a 50 ml flame dried round bottom flask with magnetic stir bar wasadded 726 mg (1.3 mmol) of2'-deoxy-5-fluoro-5'-O-(4-methoxytrityl)uridine, 32 mg (0.26 mmol) of4-dimethylaminopyridine (DMAP) and 143 mg (1.4 mmol) of succinicanhydride. The mixture was dissolved in 6 ml of pyridine sealed under aseptum and magnetically stirred overnight. The following day anadditional 30 mg of DMAP and 30 mg of succinic anhydride was added andthe reaction was stirred for an additional 24 h. On the third day, 1.05g (5.1 mmol) of 2'-deoxy-5-ethynyl-5'-O-(4-methoxytrityl)uridine wasadded and the mixture was allowed to stir overnight. The following day,dicyclohexylurea was removed via suction filtration through a sinteredglass funnel and was washed with ethyl acetate. The filtrate waspartitioned between ethyl acetate and water. The ethyl acetate layer wasdried over magnesium sulphate, filtered and evaporated on a rotaryevaporator. The title compound was obtained by successive medium airpressure (9-12 psi) silica gel (100 g, 230-400 mesh) column (2.5×30 cm)chromatography eluting with dichloromethane, followed by 1% then 3%methanol in chloroform. After evaporation of solvent, the product wasobtained as a white foam (0.27 g, 0.22 mmol): Rf 0.35 (silica, 9:1chloroform: methanol). ¹ H NMR (300 MHz, DMSO-d₆) δ 12.00-11.68 (2 bs2H, N3H & N3"H), 7.97 (s, 1H, H6), 7.89 (d, 1H, H"6, J_(F),H =6.8 Hz),7.40-7.20 (m, 10H, Ar H), 6.88 (d, 2H, ArH, J=8.8 Hz), 6.18-6,10 (m. 2H,H1' & H1'") 5.25-5.23 (m, 2H, H3' & H3'"), 4.07 (m 2H, H4' & H4'"), 4.00(s, 6H, 2 X OCH₃), 3.20-3.40 (m, obscured by water, H5' & H5'") 2.40 (s,4H, succinate), 2.50-2.20 (m, 4H, H2' & H2'").

(e)5-Ethynyl-5"-fluoro-3',3'"-O-succinylbis(1-(2-deoxy-β-D-erythro-pentofuranosyl)uracil)

0.130 g (0.11 mmol) of5-Ethynyl-5"-fluoro-3',3'"-O-succinylbis(1-(2-deoxy-5-O-(4-methoxytrityl)-β-D-erythropentofuranosyl)uracil)was dissolved in 10 ml of anhydrous methanol in a round bottom flask.400 mg of methanol-washed Dowex-50WX8 (H⁺ form, 20-50 mesh, J. T. Baker)was added and the mixture warmed to 55° C. for 4 h. The solution wasfiltered to remove the resin and the beads washed with methanol. Thefiltrate was rotary evaporated to dryness. The off-white solid wassuspended in dichloromethane and collected by suction-filtration, washedwith dichloromethane then water and dried in a vacuum oven overnight at50° C. 0.035 g (0.059 mmol) of an off-white solid was obtained. ¹ H NMR(300 MHz, DMSO-d₆) δ 12.0 (bs, 2H, N3H & N3"H), 8.29 (s, 1H, H6), 8.21(d, 1H, H"6, J_(F),H =7.1 Hz), 6.16-6.12 (m, 2H. H1' & H1'"), 5.35-5.30(m, 2H, H3' & H3'"), 5.59-5.24 (m, 2H,5'OH & 5'"OH), 4.14 (s, 1H,acetylenic H), 4.04-4.01 (m, 2H, H4' & H4'"), 3.65-3.63 (m, 4H, H5' &H5'"), 2.65 (s, 4H, succinate), 2.33-2.26 (m, 4H, H2' & H2'").

EXAMPLE 3 Preparation of5'-chloro-2',5'-Dideoxy-5-Dideoxy-5-fluorouridine2'"-Deoxy-5"-Ethynyluridine(3'-5'") Diphosphate

(a) 2'-Deoxy-5-(2-(Trimethylsilyl)Ethynyl)Uridine

The title compound was prepared according to the procedure of Morris J.Robbins and Philip J Barr, J.Org. Chem., 1983, 48, 1854-1862.

A mixture of 5-iodo-2'-deoxyuridine (U.S. Biochemical Corp., Cleveland,Ohio) (2.7 mmol), dimethylformamide (8 ml), and triethylamine (0.6 ml)was deoxygenated with a rapid stream of N₂ over 10 min in a flame dried100 ml round-bottom flask with magnetic stirring. To this deoxygenatedsolution was added copper iodide (Aldrich Chemical Co., Milwaukee, Wis.)(1.0 mmol), bis(triphenylphosphine)palladium(II)chloride (Aldrich) (0.5mmol), and (trimethyl silyl)acetylene (Aldrich) (5.4 mmol). The mixturewas allowed to magnetically stir at room temperature overnight, at whichtime there was no starting nucleoside as evidenced by TLC (Silica, 9:1chloroform:methanol). DMF was removed on a vacuum pump at ambienttemperature. The crude material was applied in chloroform to a 70 gsilica gel (230-400 mesh) column (2.5×30 cm) packed in 3% methanol inchloroform. Under medium air pressure (9-12 psi), the column was elutedwith 1 L of 3% methanol in chloroform. and then 1 L of 5% methanol inchloroform. 100 ml fractions were collected. Fractions 10-15 containedthe desired product. The product fractions were pooled and solvent wasremoved on a rotary evaporator to give the product as a hygroscopicvellow foam (2.3 mmol, 85% yield): R_(f) 0.15 (silica, 9:1 chloroform:methanol). 1 R (KBr) 2167 cm⁻¹, acetylenic C≡C stretch. ¹ H NMR (300MHz, DMSO-d6) δ 11.7-11.6 (bs, 1H, N-H), 8.26 (s,1H,H-6), 6.11-6.07 (t,1H, H1', J=6.5 Hz), 5.26-5.24 (d, 1H, 3'-OH, J=4.3 Hz), 5.13-5.09 (t,1H, 5'-OH, J=5.0 Hz), 4.25-4.20 (m - 5 Lines, 1H, H3'), 3.80-3.76 (q,1H, H4'), 3.63-3.55 (m, 2H, H5" and H5' overlapping), 2.16-2.10 (m, 2H,H2" and H2'), 0.20 (s, 9H, Si(CH₃)₃).

(b) 2'-Deoxy-5-Ethynyluridine

The title compound was prepared according to a modification of theprocedure of Morris J. Robbins and Philip J Barr, J. Org. Chem., 1983,48, 1854-1862.

A mixture of 2'-deoxy-5-(2-(trimethylsilyl)ethynyl)uridine (2.4 mmol)and tetraethyl ammonium fluoride hydrate (Aldrich Chemical Co.,Milwaukee, Wis.) (2.4 mmol) was magnetically stirred in 10 ml dryacetonitrile (Aldrich) for 1 hour. The solution was applied directly toa column (2.5×30 cm) of 100 g silica gel (230-400 mesh) packed indichloromethane. The column was eluted with 3% methanol indichloromethane to obtain pure product (1.7 mmol, 71% yield) as anoff-white solid after evaporation of solvent. 1R (KBr) 2120 cm⁻¹, C≡Cstretch. ¹ H NMR (300 MHz, DMSO-d6) δ 11.58-11.65 (bs, 1H, NH), 8.30 (s,1H,H6), 6.13-6.08 (t, 1H, H1', J=6.55 Hz), 5.26-5.24 (d, 1H, 3'-OH,J=4.3 Hz), 5.15-5.12 (t, 1H, 5'-OH, J=4.90 Hz), 4.26-4.21 (m - 5 lines,1H, H3'), 4.11 (s, 1h, acetylenic H), 3.82-3.79 (q, 1H, H4'), 3.63-3.56(m, 2H, H5" and H5'), 2.15-2.12 (m, 2H, H2" and H2').

(c) 1-(2-Deoxy-β-D-Erythro-Pentofuranosyl)-5-Ethynyluracil5'-Monophosphate and1-(2-Deoxy-β-D-Erythro-Pentofuranosyl)-5-Ethynyluracil 3',5'-Diphosphate

2'-Deoxy-5-ethynyluridine (1.7 mmol) was co-evaporated several times ina 25 ml round-bottom flask with dry acetonitrile (Aldrich Chemical Co.,Milwaukee, Wis.). The nucleoside was dissolved in 5 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (Aldrich) withmagnetic stirring. The flask was sealed with a serum stopper andthe-solution stirred in a methanol-ice bath at -12° C. for 5 minutes. 3equivalents (0.4 ml) of phosphorous oxychloride (Aldrich) from a freshlyopened ampoule was added to the nucleoside solution. After 5 minreaction time, the mixture was quenched with 5 ml of water withcontinued stirring. After 3 min, 0.5 ml of tributylamine (Aldrich) wasadded. The product mixture was diluted with water and loaded onto 80 gof DEAE Sephadex swollen in 50 mM ammonium bicarbonate. The column waseluted with an increasing gradient of aqueous ammonium bicarbonate (50mM to 500 mM) with UV detection at 280 mm. Elution flowrate was ascontrolled by Gilson Minipuls 2 peristaltic pumps (Gilson, Middleton,Wis.). The first eluting product fractions were pooled and evaporated ona rotary evaporator at 45° C. The resulting white solid wasco-evaporated multiple times with water on the rotary evaporator, thenre-dissolved in 15 ml of water, frozen and lyophilised to give 0.34 mmol(20% yield) of the 5'-monophosphate as a white monoammonium salt. HPLCwas performed on a 4.6 mm X 100 mm, 5 micron, analytical strong anionexchange column (Alltech, Deerfield, Ill.) with a linear gradient of 10mM to 1M ammonium phosphate, pH 5.5, 5% methanol over 30 min. Thisproduct had a retention time (R_(t)) of 3.4 minutes. Negative Ion FAB MS(dithioerythritol/dithiothreitol) (M-H)⁻ =331.1. ³¹ P NMR (121. 421 MHz,¹ H decoupled, 85% phosphoric acid δ=0.0, DMSO-d6) δ 0.32 (s, 1P,5'phosphate). 'H NMR (300 MHz₂, DMSO-d6) δ 7.97 (s, 1H, H6), 6.09-6.05(overlapping dd, 1H, H1'), 4.27-4.25 (bs, 1H, H3'), 4.05 (s, 1H,acetylenic H) 3.89-3.85 (bs, 1H, H4'), 3.79-3.76 (m, 2H, H5" and H5')2.15-2.05 (H2" and H2').

A second product eluted after the 5'-monophosphate. Identical fractionswere pooled, evaporated and lyophilised as before to give 0.62 mmol (37%yield) of the 3',5'-diphosphate of the starting nucleoside. AnalyticalHPLC (as above), R_(t) =8.0 min. Negative Ion FAB MS(dithioerythritol/dithiothreitol) (M-H)⁻ =411.0. ³¹ P NMR (121.421 MHz,¹ H decoupled, 85% phosphoric acid δ=0.0, DMSO-d6) δ -0.01 (s, 1P,5'phosphate), -1.07 (s, 1P, 3' phosphate). ¹ H NMR (300 MHz, DMSO-d6) δ8.00 (S, 1H, H6), 6.10-6.05 (t, 1H, H1', J=7.1 Hz), 4.76-4.74 (m -broad, 1H, H3'), 4.17-4.16 (s, 1H, H4'), 4.06 (s,1H, acetylenic H),3.90-3.78 (2 multiplets, 2H, H5" and H5'), 2.22-2.21 (m - broad, 2H, H2"and H2').

The 5'-monophosphate and the 3',5-diphosphate were assayed for ammoniaaccording to the assay of A. L. Chaney and E. P. Marbach, Clin. Chem.1962, 8, 130-132. The phosphates were assayed for phosphorous accordingto procedure of B. N. Ames, Methods Enzymol. 1966, 8, 115-118.

(d) Preparation of1-(2-Deoxy-β-D-Erythro-Pentofuranosyl)-5-Ethynyluracil 5'-MonophosphateFrom 1-(2-Deoxy-β-D-Erythro-Pentofuranosyl)-5-Ethynyl uracil3',5'-Diphosphate By Enzymatic Dephosphorylation With Nuclease P1

To 1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-ethynyluracil3',5'-diphosphate (0.50 mmoles) in 5 ml of water was added 50microliters of aqueous 1M ZnCl₂ and 250 microliters of a 30 mM sodiumacetate buffer, pH 5.0, containing 1 unit of Nuclease P1 (fromPenicillium citrinum, Boehringer Mannheim, Indianapolis, Ind.) permicroliter. The mixture was heated in a water bath at 50° C. for 3hours. The product was purified and characterised as previouslydescribed. The yield was quantitative.

(e) 5'-Chloro-2',5'-Dideoxy-5-Fluorouridine 3'-O-Phosphate and2'-Deoxy-5-Fluoro uridine 3',5'-Di-O-Phosphate

This is a modification of the procedure found in European PatentApplication No. 910502.6; Inventor, S. M. Tisdale et al.; Title,Antiviral Compounds. and Bull.Chem.Soc.Japan (1969), 42, 350.

2'-Deoxy-5-fluorouridine (4.1 mmol) (United States Biochemical,Cleveland, Ohio was dissolved in 7 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (Aldrich) in a vialequipped with a magnetic stir bar and cap. The vial was cooled to -5° C.in an ice-methanol bath. 1.5 ml of phosphorous oxychloride was added tothe stirring solution and the solution continued to stir in the bath for7 min. The reaction was then quenched by the addition of 5 ml of water,and the mixture was allowed to stir in the cold bath for 5 min. Themixture was poured into 30 ml of tributylamine, diluted with 900 mlwater and loaded onto 80 g of DEAE Sephadex swollen in 50 mM ammoniumbicarbonate. The column was eluted with an increasing gradient ofaqueous ammonium bicarbonate (50 mM to 500 mM) with UV detection at 267nm. Elution flowrate was controlled by Gilson Minipuls 2 peristalticpumps (Gilson, Middleton, Wis.). The first eluting product fractionswere pooled and evaporated on a rotary evaporator at 45° C. Theresulting white solid was co-evaporated multiple times with water on therotary evaporator, then re-dissolved in 15 ml of water, frozen andlyophilised to give 1.2 mmol (29% yield) of the5'-chloro-2',5'-dideoxy-5-fluorouridine 3'-O-phosphate as themono-ammonium salt. HPLC was performed on a 4.6 mm×100 mm, 5 micron,analytical strong anion exchange column (Alltech, Deerfield, Ill.) witha linear gradient of 10 mM to 1M ammonium phosphate, pH 5.5, 5% methanolover 30 min. Ths product had a retention (R_(t) of 3.6 minutes. PositiveIon FAB MS (glycerol) (M+1)⁺ =345.1. ³¹ P NMR (121. 421 MHz, ¹ Hdecoupled, 85% phosphoric acid δ =0.0, DMSO-d6) 67 -0.56 (s, 1P,3'-phosphate). ¹ H NMR (300 MHz, DMSO-d6) δ 7.96-7.94 (d, 1H, H6, J=7.0Hz), 6.15-6.13 (triplet - broadened, 1H, H1'), 4.56-4.54 (bs, 1H, H3'),4.17-4.16 (bs, 1H, H4'), 3.95-3.90 (overlapping multiplets, 2H, H5 " andH5', 2.39-2.19 (m, 2H, H2" and H2').

A second product eluted after the 5'-monophosphate. Identical fractionswere pooled, evaporated and lyophilised as before to give 0.62 mmol (37%yield) of the 3',5'-diphosphate of the starting nucleoside. AnalyticalHPLC (as above), R_(t) =8.5 min. Positive Ion FAB MS (glycerol) (M+1)⁺=407.4. ³¹ P NMR (121.421 MHz, ¹ H decoupled, 85% phosphoric acid δ=0.0,DMSO-d6) 67 -0.369 (s, 1P,5'phosphate), -1.45 (s, (1P, 3' phosphate). ¹H NMR (300 MHz, DMSO-d6) δ 8.12-8.10 (d, 1H, H6, J=7.0 Hz), 6.12-6.10(t, 1H, H1'), 4.82-4.79 (m, 1H, H3'), 2.23-2.20 (m, 2H, H2" and H2').

The phosphates were assayed for phosphorous according to procedure of B.N. Ames, Methods Enzymol. 1966, 8, 115-118.

(f) 5'-Chloro-2',5'-Dideoxy-5-Fluorouridine 2'"-Deoxy-5"-Ethynyluridine(3',5'") Diphosphate

This phosphoimidazolidate procedure is based on the synthesis oftriphosphates from monophosphates in the paper by Donald E. Hoard andDonald G, Ott, J. Am. Chem. Soc. 1965, 87, 1785-1788.

The 5'monophosphate of 1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-ethynyluracil (0.61 mmol) was co-evaporated with 2×50 ml 0.5M triethylammoniumbicarbonate at 40° C. on a rotary evaporator to a tan foam. Thenucleotide was then co-evaporated with 3×75 ml of dry acetonitrile(Aldrich Chemical Co., Milwaukee, Wis.) in the same round-bottom flask.The nucleotide thus dried was dissolved in 5 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (Aldrich) withmagnetic stirring. Approximately 5 equivalents (498 mg) of1,1'-carbonyldiimidazole was added to the solution and the mixturestirred for 1 h with the flask sealed with a serum stopper. At thistime, 157 microliters of methanol was added and the solution stirred for75 min to destroy excess 1,1'-carbonyldiimidazole.

1.2 Equivalents of 5'-chloro-2',5'-dideoxy-5-fluorouridine3'-O-phosphate was co-evaporated with 0.5M triethylammonium bicarbonate,then dry acetonitrile as the previous nucleoside. The 5-fluoronucleotide was then dissolved in 4 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone and added to the5-ethynyl nucleotide phosphorimidazolidate mixture. This new mixture wasallowed to stir overnight. The following day an additional 0.25equivalent of the 5-fluoro nucleotide, similarly prepared, was added tothe phosphorimidazolidate mixture and the resulting solution stirredover the weekend. This condensation mixture was then diluted into 150 mlaqueous ammonium hydroxide, pH 9.5; 500 microliters of alkalinephosphatase (Boehringer Mannheim, Indianapolis, Ind.) was added tocleave non-coupled nucleotides and the mixture was allowed to stirovernight. The following day, the alkaline solution was diluted to 600ml total volume with water and loaded onto 80 g of DEAE Sephadex swollenin 50 mM ammonium bicarbonate. The column was eluted with an increasinggradient of aqueous ammonium bicarbonate (50 mM to 500 mM) with UVdetection at 270 nm. Elution flowrate was controlled by Gilson Minipuls2 peristaltic pumps (Gilson, Middleton, Wis.). Fractions were analysedby HPLC with a 4.6 mm×100 mm, 5 rnicron, analytical strong anionexchange column (Alltech, Deerfield, Ill.) and a linear gradient of 10mM to 1M ammonium phosphate, pH 2.4, 5% methanol over 30 min. Pureproduct fractions were pooled and water was removed on a rotaryevaporator. The resulting white solid was co-evaporated multiple timeswith water on the rotary evaporator, then re-dissolved in 15 ml ofwater, frozen and lyophilised to give 0.18 mmoles (30% yield) of thetitle product. Analytical HPLC (as above), R_(t) =5.62 min. Negative IonFAB MS (dithioerythritol/dithiothreitol) (M-H)⁻ =657.0. ³¹ P NMR (1 MHz,¹ H decoupled, 85% phosphoric acid δ=0.0, D₂ O) δ (-) 10.14 - (-) 10.27(d, 1P, 5'-Phosphate, J_(p),p =21.48 Hz), (-) 10.94 - (-) 11.08 (d, 1P,3'-Phosphate, J=21.48 Hz) (³¹ P assignments made by ¹ H - ³¹ P HMQC NMRpulse sequence). ¹ H NMR (400 MHz, D₂ O) δ 8.05 (s, 1H, H6"), 7.80 (d,1H, H-5, J=7.0 Hz) 6.17-6.15 (m - overlapping triplets, 2H, H1', andH1'"), 4.78-4.74 (m, 1H, H3'), 4.42-4.40(m - 5 lines, H3"), 4.32-4.28(m, 1H, H4'), 4.08-4.04 (m, 1H, H4'"), 4.03-4.00 (m, 2H, H5'"a andH5'"b), 3.76-3.73 (m, 2H, H5'a and H5'b), 3.44 (s, 1H, acetylenic H),2.50-2.20 (m overlapping, 4H, H2'a, H2'b, H2'"a and H2'"b).

The product was assayed for phosphorous according to procedure of B. N.Ames, Methods Enzymol. 1966, 8, 115-118.

EXAMPLE 4 Method

(a) Animal Dosing and 5-FU Urinary Sampling

5-FU and 5-EU bases and nucleosides were dissolved in deionized HPLCgrade water immediately prior to dosing. All animals were dosed once,p.o. via gastric gavage at the indicated mg/kg dose for each compound(See Results Section and Figures). 5-EU and 5-EU nucleosides were dosedto animals 0.5 hr prior to dosing with 5-FU and 5-FU nucleosides. Dosingwas routinely performed between 11 am and 1 pm. An approximate volume of300 μL was used for each dose. Rats were placed in individual Nalgemetabolism cages, which separate urine and faeces, and continued on a 12hr light/dark schedule post-dosing. Mice were treated post-dose in asimilar manner with the exception that 5 mice were housed per cage.

Urine from each cage was collected into 0-24 hr and 24-48 hr post-dosefractions. Cages were rinsed with deionized water prior to collection ofeach fraction. The combined volume of urine and rinse was brought to astandard volume with deionized water.

(b) Quantitation of 5-FU by Reversed-Phase HPLC Analysis.

5 ml of a thoroughly mixed urine sample was filtered through a Millex GS0.22 μm filter (Millipore, Bedford, Mass.) and frozen prior to HPLCanalysis. 1 ml of the thawed primary filtered solution was diluted with2 ml of water and particulates were removed from this diluted stocksolution using a Centrifree micro partition system (Amicon Division, W.R. Grace and Co., Beverly Mass.). 100 μL of this solution wasquantitated for 5-FU by HPLC on a Waters Associates System (WatersAssociates, Milford, Mass.) equipped with a Waters 600E systemcontroller, a Waters 600 solvent delivery system, a Waters model 712WISP automated sample injector, a Waters 484 tuneable absorbancedetector, and a Waters 991 photodiode array detector. For quantitiationof 5-FU in rat urine, HPLC was performed on a 250 mm×4.6 mm YMC AQ-303S-5 120A ODS analytical column (YMC, Morris Plains, N.J.) or on aMicrosorb C18 analytical column with identical dimensions (RaininInstrument Co., Woburn, Mass.). For each analysis the columns were firsteluted isocratic at 0.5 ml/min with aqueous 50 mM ammonium acetatebuffer, pH 4.8, and 0.5% acetonitrile (Buffer A) for 25 min, followed bya linear gradient over 15 min to a 50% mixture with aqueous 50 mMammonium acetate buffer, pH 4.8, and 60% acetonitrile (Buffer B). Overthe next 5 min a linear gradient was run to 100% buffer B, followed byisocratic elution with buffer B for 20 min. A linear gradient was theneluted across the column over a 10 min period to 100% buffer A and heldat a flow rate of 0.5 ml for 2 min. The column was then re-equilibratedin buffer A at a flow rate of 0.75 ml/min for 15 min and then returnedto the original flow rate (0.5 ml/min) for 2 min prior to injection ofthe next sample. The effluent was monitored at 266 nm and 5-FU had aretention time of ca. 16.5 min. Data was collected on the Waters 991diode array detector and a Digital Specialties microcomputer equippedwith an in-house HPLC data analysis program (CHROM). The UV peak areaswere integrated on these systems and compared to those of a standardcurve prepared from known aqueous concentrations of 5-FU isocraticallyeluted across the Microsorb column with Buffer A. The molar extinctioncoefficient used for 5-FU was 7070 M⁻¹ cm⁻¹ at a λmax of 266 nm.

Plots of UV peak area vs 5-FU concentration were linear between 5 μM and100 μM. The total urinary recovery of 5-FU was determined bymultiplication of the nmoles of 5-FU determined from the standard curveby the dilution factor of 3 (above) and by the standard volume of urinecollected.

5-FU recovery in mouse urine was determined as above with threeexceptions: a Phenomenex 5 μm Extrasil C-18 column (250mm×4.6 mm i.d.;Phenomenex, Torrance, Calif.) was used; an identical gradient withbuffer A as aqueous 50 mM formic acid, pH 3.5, with 0.5% acetonitrileand buffer B as aqueous 50 mM formic acid, pH 3.5, with 60% acetonitrilewas used; the standard curve was prepared by diluting knownconcentrations of 5-FU into undosed mouse urine and eluting with thelatter described gradient and buffers, instead of isocratic conditions.

Results

(a) Release and Quantification of 5-FU From Nucleosides.

Rats were predosed with 5-EU (2 mg/kg, p.o.) 0.5 hr prior to dosing withthe 5-FU nucleosides as shown in Table 1. The 5-FU nucleosides weredosed at either 10 or 25 mg/kg. The arabinoside and2',3'-dideoxyriboside required the higher dosing in order to detect andquantify urinary recovery of 5-FU. For all nucleoside examples, 5-FU wasonly detected in the 0-24 hr urine and not in the 24-48 hr urinesampling. The riboside and 2'-deoxyriboside are optimum in vivoreleasers of 5-FU with a urinary recovery of 5-FU>65%.

                  TABLE 1                                                         ______________________________________                                        Urinary Recovery of 5-FU In Rats For                                          Various 5-FU Containing Nucleosides                                                                     % 5-FU Recovery                                                               0-24 hr Urine                                                 Dose            (Average of Two                                               (mg/kg) Predose Experiments)                                        ______________________________________                                        5-FU         5        --      <5                                              5-FU         5        2       52                                              5-FU        25        0.03    43-63                                           5-FU Nucleosides                                                              Riboside    10        2       78                                              2'-deoxyriboside                                                                          10        2       67                                              Tegafur     10        2       38                                              5'-chloro-2',5'-                                                                          50        0.03    21                                              dideoxyriboside                                                               Arabinoside 25        2        6                                              2',3'-dideoxy         2       <5                                              riboside                                                                      ______________________________________                                    

(b) Protection of 5-FU From DPD Catabolism by Release of 5-EU FromNucleosides.

As seen in FIG. 1, rats were orally predosed with 0.03 mg/kg 5-EU andwith 0.03, 0.3 and 3.0 mg/kg of various 5-EU nucleosides, prior todosing with 25 mg/kg 5-FU. 5-FU was observed and quantitated in only the0-24 hr urine samples. Approximately 65% of the 5-FU dose was recoveredunchanged in the urine for the 5-EU treated rats. Predosing rats witheither the riboside or the 2'-deoxyriboside of 5-EU (≧0.03 mg/kg)allowed for high urinary recovery (>40%) of 5-FU. A direct dose responsefor urinary recovery of 5-FU was observed when the arabinoside or the2'-deoxy-5'-monophosphate of 5-EU was used to deliver the DPDinactivator. Urinary recovery of 5-FU was <5% at all predoses of2',3'-dideoxy-5-ethynyluridine.

(c) Urinary Recovery of 5-FU in Rats Dosed with the Succinate LinkedNucleoside

Rats were dosed 25 mg/kg 5-FU equivalent with the succinate linkednucleosides. Greater than 35% of the 5-FU in the original dose wasrecovered unchanged in the 0-24 hr urine. 5-FU was not detected in the24-48 hr urine.

(d) Urinary Recovery of 5-FU in Mice Dosed with the Succinate linkedNucleosides.

For these studies mice were dosed with either 5 mg/kg 5-FU or given anequivalent dose of 5-FU via the succinate linked nucleoside. The urinaryrecovery of 5-FU was measured in the presence and absence of 5-EU (Table2). Without predosing mice with 5-EU before delivering the base 5-FU,5-FU could not be detected in the urine. When mice were predosed with5-EU followed by dosing with 5-FU, the urinary recovery of 5-FU was 50%.Mice dosed with the succinate linked nucleoside gave 25-30% urinaryrecovery of 5-FU available from the linked compound. There was noimprovement of 5-FU urinary recovery from mice that were predosed with5-EU followed by dosing with the succinate linked nucleoside.

                  TABLE 2                                                         ______________________________________                                                                       Recovery                                       Dose 5 mg/kg equivalent 5-FU                                                                         Predose 5-FU (%)                                       ______________________________________                                        5-FU                   --       0                                             5-FU                   2 mg/kg 51                                             5-Ethynyl-5"-fluoro-3,3'''-O-succinylbis(1-(2-                                                       --      28                                             deoxy-β-D-erythropentofuranosyl)uracil                                   5-Ethynyl-5"-fluoro-3,3'''-O-succinylbis(1-(2-                                                       2 mg/kg 23                                             deoxy-β-D-erythropentofuranosyl)uracil                                   ______________________________________                                    

EXAMPLE 5 Antitumour Studies Method

In vivo antitumor testing with murine Colon 38 was conducted in-house.C57BL/6 female mice were housed in sterile, polycarbonate, filter-cappedMicroisolator cages (Lab Products, Inc., Maywood, N.J.) containingsterile hardwood bedding. Mice were fed sterile rodent chow andfilter-sterilised water ad libitum. All manipulations of these mice wereconducted in laminar-flow biosafety hoods. Colon 38 carcinoma wasobtained from the Development Therapeutics Program Tumour Repository.Mice were implanted s.c. with 70 mg tumour fragments using a number 13trocar. There were 10 mice in the control group that received tumourimplants without drug treatment. There were 5 mice in each group treatedwith the dose and compounds as described below. Treatment began on day16 post tumour implant and compounds were dosed once/day for 9 days.5-FU was dosed at 15, 20, 25, 30, and 40 mg/kg.. The succinate linkednucleoside was dosed at 4.5, 8.9, 17.9, 26.8, 35.7 and 44.6 mg/kg. Themolecular weight of the succinate linked nucleoside is 580.40 and thecorresponding equivalents of 5-FU that were dosed with this compoundwere 1.0, 2.0,4.0, 6.0, 9.0 and 10.0 mg/kg. A sample of 5-FU and thesuccinate linked nucleoside doses are indicated in Table 3. Tumourweights were calculated three times per week from measurements of tumourlength and width. Antitumor activity was expressed as the days delay intumour growth (T-C). T-C was calculated as the difference in the medianof days for treated animals minus control animals for tumour mass todouble twice post initial day of dosing (Day 16). Tumour-free survivorswere not excluded from T-C calculations. Surviving mice were sacrificedafter day 56.

Results

Antitumor Efficacy of5-ethynyl-5"-fluoro-3.3'"-O-succinylbis(1-(2-deoxy-β-D-erythropentofuranosyl)uracil

The effect of the succinate linked nucleoside was studied in mice withs.c. implants of Colon 38 vs therapy with 5-FU alone. Antitumor activitywas expressed as tumour growth delay (T-C; see Methods). The succinatelinked nucleoside was toxic at doses >17.9 mg/kg (4 mg/kg (4 mg/kg 5-FUand 5-EU equivalents). Table 3 shows a sub-optimum, effective and toxicdose for 5-FU and the succinate linked nucleoside.

                  TABLE 3                                                         ______________________________________                                                  Dose        Antitumour                                                                              Tumour-free                                             (mg/kg 5-FU Activity T-C                                                                            Survivors                                     Compound  equivalent) (Days)    (Day 56)                                      ______________________________________                                        5-FU      25          14.9      0/5                                           5-FU      30          23.6      0/5                                           5-FU      40          Toxic     0/5                                           *          1          12.9      0/5                                           *          2          >27.9     2/5                                           ______________________________________                                         *.tbd.5Ethynyl-5fluoro-3,3''O-succinylbis(1-(2-deoxy-D-erythropentofurano    yl)uracil)                                                                

We claim:
 1. A compound selectedfrom:5-ethynyl-5"-fluoro-3',3'"-O-succinylbis-(1-(2-deoxy-β-D-erythropentofuranosyl)uracil);5-ethynyluridine-5-fluoro-1-(β-D-arabinofuranosyl)uracil succinic acid5',2'-diester;5-fluoro-5"-(1-propynyl)-2',2'"-O-succinylbis(1-β-D-arabinofuranosyl)uracil);and 5-chloro-2'-5'-dideoxy-5-fluorouridine-2'"-deoxy-5"-ethynyluridine(3'-5'")diphosphate.
 2. A pharmaceutical composition comprising acompound selectedfrom:5-ethynyl-5"-fluoro-3',3'"-O-succinylbis-(1-(2-deoxy-β-D-erythropentofuranosyl)uracil);5-ethynyluridine-5-fluoro-1-(β-D-arabinofuranosyl)uracil succinic acid5',2'-diester;5-fluoro-5"-(1-propynyl)-2',2'"-O-succinylbis(1-β-D-arabinofuranosyl)uracil);and 5-chloro-2'-5'-dideoxy5-fluorouridine 2'"-deoxy-5'"-ethynyluridine(3'-5'")diphosphatetogether with one or more pharmaceutically acceptablecarriers.
 3. A pharmaceutical composition according to claim 2 for oraladministration.